Luminescent nanostructured materials for use in electrogenerated chemiluminescence

ABSTRACT

A nanostructured particulate material, which includes a redox active luminescent organic and/or ionic compound, is provided herein. The nanostructured particulate material may be used for determining the presence of an analyte of interest in a sample by detecting the emitted electromagnetic radiation generated by exposing a reagent mixture, which includes the nanostructured material and the target analyte, to chemical or electrochemical energy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a national stage of International PatentApplication No. PCT/US2009/002534, filed on Apr. 24, 2009; and claimsthe benefit of U.S. Provisional Patent Application 61/126,892, filed onMay 8, 2008; and U.S. Provisional Patent Application 61/127,311, filedon May 12, 2008; the entire contents of which are hereby incorporated byreference, for any and all purposes.

BACKGROUND

Nanoparticles (“NPs”) have been reported to have a wide range ofapplications in electronics, optics, catalysis and biotechnology. Thephysical properties (e.g., high surface-to-volume ratio, elevatedsurface energy, increased ductility after pressure loading, higherhardness, larger specific heat and the like) of NPs have led to avariety of applications in the material-directed industry and materialscience. For example, a variety of metal NPs have been used to catalyzenumerous reactions and semiconductor NPs are used as fluorescent probes.

Single particle electrochemical sensors, which employ an electrochemicaldevice for detecting single particles, have also been reported. Methodsfor using such a device to achieve high sensitivity for detectingparticles such as bacteria, viruses, aggregates, immuno-complexes,molecules, or ionic species have been described.

The use of colloidal particles in sensing arrays have also beenreported. These are chemical sensors for detecting analytes in fluidsvia arrays having a plurality of alternating nonconductive regions andconductive regions of conductive NP materials. Variability in chemicalsensitivity from sensor to sensor is reported to be provided byqualitatively or quantitatively varying the composition of theconductive and/or nonconductive regions.

The size of nanostructured materials (“NSMs”) generally ranges from lessthan 1 nm to several hundred nm at least in one dimension and theelectronic energy band configuration is a size-dependent property, whichin turn can affect the physical and chemical properties. A fundamentaldistinction between NSMs and bulk materials is that the fraction ofsurface atoms and the radius of curvature of the surface of NSMs arecomparable with the lattice constant. As a result, nanostructuredmaterials generally have higher activity as compared with theiranalogues based on bulk materials. A number of methods of forming NSMsare known to the skilled artisan and include formation by combiningatoms (or more complex radicals and molecules) and by dispersion of bulkmaterials, e.g., thermal evaporation, ion sputtering, reduction fromsolution, reduction in microemulsions, and condensation.

SUMMARY

The present application relates, in general, to the field ofnanostructured materials, such as nanoparticles (NPs), including thesynthesis and characterization of organic and/or ionic luminescentnanostructured materials. Such nanostructured materials (“NSMs”) formedfrom luminescent organic and/or ionic materials may be employed forelectrogenerated chemiluminescence (ECL). The difficulties ingenerating, locating and characterizing NPs, especially at the nm scaleand in measuring the very small current and ECL intensity generated bythe electrode reactions at NPs has been recognized. The presentapplication provides a method and apparatus, which can be used forobserving the ECL generated during the collisions of nanostructuredmaterials at the electrode. The present method can provide informationwith respect to the electrochemical processes of the nanostructuredmaterials, as well as provide the basis for highly sensitiveelectroanalytical methods. The electrochemical properties measured inthe present method can be any property that can be measured by theapparatus; however, the most common property involves ECL generationfrom a redox reaction of the nanostructured materials. Another commonlymonitored property can be a current.

The present device commonly includes an electrochemical cell in a samplechamber. The electrochemical cell typically has two or more electrodes,one or more ports for introducing nanostructured materials into thesample chamber, and an electrochemical apparatus in communication withthe electrodes. The electrochemical cell may be connected to a measuringapparatus which includes an electrochemical apparatus and a photondetector. The injected nanostructured materials can interact with theelectrode and generate one or more photons that can be picked up by aphoton detector.

The present invention includes a kit for analyzing one or more chemicalanalyte(s) having at least one ECL NSM, at least two electrodes, anoptional co-reactant and a measuring apparatus that reads one or more ofcurrent and ECL properties generated by the interactions between theNSM(s), the electrode(s) and the chemical analyte(s).

One embodiment provides a method for detecting an analyte in a samplesolution to which luminescent nanostructured materials have been added.The method includes introducing the sample solution into anelectrochemical cell containing two or more electrodes in communicationwith the solution; generating one or more ECL properties through aninteraction of the luminescent nanostructured materials, the liquidsample and one or more of the electrodes; and measuring at least one ECLproperty generated by the interaction. The luminescent nanostructuredmaterials include a redox active, luminescent organic and/or ioniccompound. A coreactant may be added to the liquid sample to enhance thegeneration of the ECL properties. Examples of suitable coreactantsinclude oxalate salts (e.g., sodium oxalate), persulfate, benzoylperoxide and trialkyl amines (e.g., tripropyl amine).

One embodiment provides a method for detecting an analyte in a samplesolution to which a plurality of luminescent nanostructured materialshave been added. The method includes introducing the sample solutioninto an electrochemical cell containing two or more electrodes incommunication with the solution; generating one or more ECL propertiesthrough an interaction of the luminescent nanostructured materials, theliquid sample and one or more of the electrodes; and measuring at leastone ECL property generated by the interaction. The luminescentnanostructured materials include a redox active, luminescent organicand/or ionic compound.

Another embodiment provides a method for observing interaction of ananostructured material with an electrode surface comprising:

contacting a dispersion of luminescent nanostructured materials in aliquid sample with one or more electrodes;

exposing the dispersion to electrochemical energy through the one ormore electrodes;

measuring at least one ECL property generated by an interaction of aluminescent nanostructured material with one of the electrodes.Measuring the ECL property(s) may include measuring an electrochemicalproperty, such as a current, generated by the interaction. Measuring theECL property(s) may include measuring an optical property, such as ECL,generated by the interaction. Measuring the ECL property(s) may includemeasuring one or more ECL properties generated by an interaction of aluminescent nanostructured material with a surface of one of theelectrodes. The dispersion is typically an aqueous colloid solution ofnanostructured materials, which are formed from a redox active,luminescent organic or organometallic compound.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent compositions, methods and devices, reference is now made to thedetailed description there along with the accompanying FIGS. and inwhich:

FIG. 1 shows an SEM image of 9,10-diphenylanthracene (“DPA”) nanorods.

FIG. 2A shows an SEM image of Ru-LCE nanobelts (“NBs”) and FIG. 2B showsa TEM image of Ru-LCE nanobelts (“NBs”). Insets in FIG. 2A and FIG. 2Bshow side-face SEM image and SAED pattern of single NB, respectively.

FIG. 3A, FIG. 3C, and FIG. 3D show TEM images and FIG. 3B shows SAEDpattern of Ru-LCE samples obtained at room temperature in early stagesafter re-precipitation. (FIG. 3A and FIG. 3B) 5 min. (FIG. 3C) 30 min.(FIG. 3D) 2 hours. ([Ru-LCE]=1.35×10⁻⁵ M, pH 7.0).

FIG. 4A shows normalized absorption (dashed) and fluorescence emission(solid) spectra of Ru-LCE NBs (“NBs-Ab” and “NBs-Em” respectively) inwater and normalized absorption (dashed) and fluorescence emission(solid) spectra of Ru-LCE monomers in MeCN (i.e., Ru-LCE molecules insolution; “Mono-Ab” and “Mono-Em” respectively). Concentrations ofRu-LCE monomers and NBs are 6.75×10⁻⁶ M and 2.25×10⁻¹⁴ M, respectively.FIG. 4B is fluorescence image of single NB.

FIG. 5A shows cyclic voltammogram (“CV”) and ECL curve of Ru-LCE NBsuspension; FIG. 5B shows current and ECL for potential steps between−1.25 V and 1.25 V. Step durations are 2, 6, 10, 14 and 18 seconds ateach potential. Supporting electrolyte and co-reactant are 0.1 Mphosphate buffer solution (pH 7.2) and 0.1 M tripropyl amine (“TPrA”),respectively. Concentration of NBs is 2.2×10⁻¹⁴ M. Platinum electrode,1.5 mm, (FIG. 5A) potential scan rate, 0.1 V/s and (FIG. 5B) pulsewidth, 2 seconds.

FIG. 6A shows CV and ECL curve of single Ru-LC NB deposited on aplatinum ultramicroelectrode (UME) in 0.1 M phosphate buffer solution(pH 7.2) containing 0.1 M TPrA. Potential scan rate, 0.1 V/s. FIG. 6B isoptical microscope image of UME with single NB.

FIG. 7A shows chronoamperometry (dotted line) and transient ECL (solidline) for rubrene nanocrystals (NCs) (prepared from THF), 0.1 M TPrA,0.1 M NaClO₄, pulse width 0.1 s (FIG. 7B) CV, sweeping ECL of rubreneNCs, blank experiment; 0.1 M TPrA, 0.1 M NaClO₄, scan rate: 500 mV/s.FIG. 7C shows chronoamperometry (dotted line) and transient ECL (solidline) for rubrene NCs (prepared from DMF), 0.1 M TPrA, 0.1 M NaClO₄,pulse width 0.1 s (FIG. 7D) Transient ECL (solid line) for rubrene NCs(prepared from THF), 0.1 M TPrA, 0.1 M NaClO₄, pulse width 0.05 second.

FIG. 8 shows ECL (solid line) and current (dotted line) of DPA nanorodsin aqueous solution containing 0.1 M Na₂C₂O₄ (sodium oxalate) as theco-reactant.

DETAILED DESCRIPTION

Methods, apparatus and kits for analyzing a chemical analyte using anelectrochemical cell connected to a measuring apparatus are providedherein. The electrochemical cell contains a solution which includes oneor more redox active luminescent nanostructured materials, one or morechemical analytes, and optionally, coreactant. In addition, theelectrochemical cell contains two or more electrodes in electricalcommunication with the solution. Two or more ECL properties aregenerated by the interaction of the luminescent nanostructured materialsand the liquid sample and measured at one or more of the electrodes.

By modifying the particle concentration, particle size and concentrationof the co-reactant (if a co-reactant is used), i-t profiles and/or ECLintensity vs. time curves may be used to obtain information about thereaction kinetics of indicator and co-reactant. In comparison tooptical, conductivity and mass signals using nanostructured materials,the present nanoparticle-based ECL technique can permit detection with asimple apparatus at high sensitivity.

The present application includes methods, compositions and kits foranalyzing a chemical analyte having an electrochemical cell connected toa measuring apparatus. The electrochemical cell contains a solutionhaving one or more nanostructured materials, with or withoutco-reactant. In addition, the electrochemical cell contains two or moreelectrodes in communication with the solution. One or more emissionevents are generated by the interaction of the one or morenanostructured materials and the co-reactant (if the co-reactant ispresent) and measured at the electrodes or an optical detector connectedto the cell.

The present application provides devices which may include one or moreredox active luminescent nanostructured materials in solution within theelectrochemical cell. For example, the one or more redox activeluminescent nanostructured materials may be ionic nanostructuredmaterials (e.g., formed from a organometallic compound) ornanostructured materials of small organic compounds or polymers. Thenanostructured materials may be of a size between about 1 nm and lessthan about 1000 nm, at least in one dimension. Furthermore, the sizedistribution of nanostructured material may be generally uniform,disperse, or varying. The nanostructured materials may have differentgroups of particles that have generally similar size within the groupbut differing size relative to other groups in the solution. Forexample, in many embodiments the nanostructured particulates have aleast one dimension which has an average size no larger than about 250nm and, in some instances, no larger than about 100 nm. Thenanostructured particulates may be of a size and shape such that noaverage dimension is larger than about 500 nm.

The present application provides methods for the preparation ofnanostructured materials including organic and/or ionic luminescentcompounds. Examples of suitable organic luminescent compounds mayinclude luminescent aromatic compounds, e.g., luminescent polycyclicaromatic hydrocarbons. Examples of suitable ionic luminescent compoundswhich may be employed in the present methods include luminescentmetal-containing complexes, e.g., polydentate complexes of a metal ion.Suitable metals which may be included in such compounds includeruthenium, osmium, rhenium, iridium, platinum, cerium, europium,terbium, and/or ytterbium. Ruthenium-containing organometallic compoundsare commonly employed in the present nanostructured materials andmethods. The methods also include embodiments, where the nanostructuredmaterial includes luminescent nanoparticles formed from luminescentphenyl substituted, polycyclic aromatic hydrocarbons, such as rubreneand diphenylanthracene (DPA).

As used herein, the phrase “nanostructured material” refers to materialsthat have a bulk structure on the nano-scale, i.e., have at least onedimension which is no larger than about 250 nm. In other words, when thematerials are in the solid state, crystals or materials of givenstructure are formed from the compounds that comprise the bulk material.Nanostructured materials, as used herein, are not individual compounds.

The metal-containing organic compound includes polydentate ligands,e.g., heteroaromatic polydentate ligands such as bipyridyl, substitutedbipyridyl, 1,10-phenanthroline and/or substituted 1,10-phenanthroline,where one or more of the polydentate ligands includes at least one longchain hydrocarbon group, e.g., a linear long chain alkyl group typicallyhaving from 12 to 22 carbon atoms. For example, the metal-containingorganic compounds include compounds of the formula[M(PD)₂(C(O)O(CH₂)_(n)CH₃)₂-PD)]X₂, where PD is a polydentate ligand; Mis a metal ion, such as a Ru or Os ion; n is an integer from 10 to 30;and X is an anion. Exemplary compounds where M is Ru and PD is abipyridyl (bpy) group, may have the following formula, referred toRu-LCE, which refers to a ruthenium compound having a long chain alkylgroup attached as an ester, hence the term LCE (long chain ester):

Specific examples of suitable ECL moieties include compounds whichinclude at least one long chain alkyl substitutedbis(2,2′-bipyridyl)ruthenium(II) or tris(2,2′-bipyridyl)ruthenium(II)moiety. One group of such compounds which can act as an ECL label arelong chain alkyl substituted Ru(bpy)₃ ²⁺ salts, e.g.,Ru(bpy)₂(4,4′-(C(O)O(CH₂)_(n)CH₃)₂-bpy)Cl₂ andRu(bpy)₂(4,4′-(C(O)O(CH₂)_(n)CH₃)₂-bpy)(ClO₄)₂, where n is an integerfrom 10 to 30. Specific examples includeRu(bpy)₂(4,4′(C(O)O(CH₂)₁₄CH₃)₂-bpy)²⁺ salts (also referred to herein as“Ru(bpy)₂(bpy-C₁₆ESt)²⁺ salts”).

Other suitable examples of long chain alkyl substituted Ru(bpy)₃ ²⁺salts include compounds such as Ru(bpy)₂(LCsub-bpy)²⁺ salts, where the“LCsub-bpy” ligand is a long chain alkyl substituted bipyridyl compound.Long chain alkyl substituted bipyridyl compounds can be prepared by anumber of methods known to those of skill in the art. Examples includethe product of reaction of metalbis(2,2′-bipyridine)(4-methyl-4′-aminomethyl-2,2′-bipyridine) salts withan activated carboxylic acid, such as stearoyl chloride or otheractivated long chain alkanoic acid derivative (e.g., CH₃(CH₂)_(n)CO₂—X,where n is an integer from about 10 to 25), to form the correspondingdiamide. Ru(II)bis(2,2′-bipyridine)(4-methyl-4′-aminomethyl-2,2′-bipyridine salts arealso referred to herein as “Ru(bpy)₂(bpy-C₁₉Amd)²⁺ salts”. Examples ofsynthetic methods to produce long chain alkyl substituted bipyridinesare described in U.S. Pat. Nos. 5,324,457 and 6,808,939 and Fraser etal., J. Org. Chem., 62, 9314-9317 (1997), the disclosures of which areherein incorporated by reference.

Long chain alkoxy substituted bipyridines can be produced by reaction ofa long chain alkoxide (e.g., NaO(CH₂)_(n)CH₃, where n is an integer fromabout 10 to 25) with 4,4′-bis-bromomethylbipyridine.

Long chain alkyl mercaptan substituted bipyridines can be produced byreaction of a long chain alkyl mercaptan (e.g., NaS(CH₂)_(n)CH₃ where nis an integer from about 10 to 25) with 4,4′-bis-bromomethylbipyridine.

Long chain alkoxy substituted bipyridines can be produced by reaction ofa long chain alkyl halide (e.g., Br(CH₂)_(n)CH₃, where n is an integerfrom about 10 to 25.) with 4,4′-bis-hydroxymethyl-2,2′-bipyridine. Forexample, see U.S. Pat. No. 6,808,939.

Long chain alkyl ester substituted bipyridines can be produced byesterification of 4-(4-methyl-2,2-bipyridine-4′-yl)-butyric acid with along chain alkanol (e.g., HO(CH₂)_(n)CH₃ where n is an integer from 6 to25). For example, see U.S. Pat. No. 6,808,939.

Long chain alkyl ester substituted bipyridines can also be produced byesterification of 4,4′-bis-(carboxy)bipyridine with long chain fattyalcohols, such as stearyl alcohol.

The metal-containing organic complexes including polydentate ligands(e.g., bipyridyl ligands) described above can include one or more of anumber of different metal ions so long as the complex is luminescent. Asnoted above, examples of suitable metal ions which may be employed insuch complexes include ruthenium, osmium, rhenium, cerium, europium,terbium, and/or ytterbium ions. Such compounds may variously be known ascoordination compounds or organometallic compounds. As used herein,organometallic compounds are those compounds having a metal and anorganic group, although no direct metal-carbon bond may be present inthe complex, although “organometallic” also refers to compounds with ametal-carbon bond. Coordination compounds are well known to those ofskill in the art.

The nanoparticles employed in the methods described herein can beproduced by a variety of methods known to those of skill in the art. Forexample, nanoscale structures of organic and/or ionic luminescentcompounds may be produced from a solution of a luminescent compound in asuitable solvent for the compound, and then, typically under vigorousmixing, adding the first solution into an anti-solvent for the compound.The first solution may be injected rapidly or added in a dropwise mannerin to the anti-solvent. This “re-precipitation method” may be conductedwith or without the presence of a capping agent, such as a low molecularweight surfactant, e.g., Triton X-100, a neutral charge polymer or acharged polymer. For certain embodiments, it may be advantageous to formnanoparticles by introducing a solution a solution of the luminescentcompound in an organic solvent into an aqueous solution that is free ofany added surfactant. The presence of surfactant may have a negativeeffect on the generation of ECL using the resultant nanoparticles,possibly due to the presence of a layer of surfactant on the outside ofthe nanoparticles. As employed herein, the term “substantially free ofsurfactant” refers to nanoparticles that have been prepared from amixture of organic solvent and aqueous solution that contains no addedsurfactant.

Specific examples of the production of nanoscale structures of organiccompounds formed by re-precipitation methods are described in Kasai etal., Jpn. J. Appl. Phys. (1992), 31, 1132, the disclosure of which isherein incorporated by reference. Examples of suitable solvents whichmay be employed in such re-precipitation methods include polar, watermiscible organic solvents such as acetonitrile (MeCN), acetone (MeCOMe),tetrahydrofuran (THF), N,N-dimethylformamide (DMF) and the like. Wateris commonly employed as the poor solvent in forming the presentnanostructured materials, although other solvents, e.g., hexane, canalso be employed as the poor solvent.

Without being bound by theory, it is believed that the general schemedepicted in Scheme I below provides an illustration of the basicmechanism by which an emitting excited state of anelectrochemiluminescent moiety is generated. As depicted, radicalspecies having one more or one less electron (in reference to theirnormal state) are generated and subsequently can combine to create anexcited state of the chemiluminescent moiety.

Ru-LCE nanobelts (NBs) were synthesized by a simple re-precipitationmethod from a 4% w/v solution in MeCN at room temperature. Such Ru-LCENBs are typically insoluble in water, but are soluble in polar organicsolvents that are miscible with water. For example,[Ru(bpy)₂(4,4′-(C(O)O(CH₂)₁₄CH₃)₂-bpy)]²⁺ is insoluble in water, but isvery soluble in either acetonitrile or acetone. In a typicalpreparation, 4 μL of this solution was rapidly injected into 10 mL ofhighly pure (Millipore) water under ultrasonic agitation at roomtemperature for 30 s, followed by aging in a closed vial at roomtemperature for 24 h. The resulting colloid solution is a transparentorange-yellowish solution that exhibits strong light scattering,confirming the formation of nanoparticles. With increasing aging time (amonth), a small amount of orange-yellow nanobelt precipitate settled.The nanobelts can easily be dispersed and a clear solution can bere-obtained by slight agitation.

SEM and TEM images, as shown in FIG. 2A and FIG. 2B, indicate that theparticles obtained after long time aging have a long, straight,belt-like morphology with widths of about 200 to 1000 nm and lengths ofabout 5 to 15 μm. FESEM (field-emission scanning electron microscopy)may also be used to characterize the NBs. The thickness of the NBsranges from around 50 to 120 nm, as estimated from the side-face SEMimage of NBs and the width-to-thickness ratios are about 5 to 10. Theselected-area electron diffraction (SAED) pattern (inset in FIG. 2B)reveals that the as-prepared NBs have single crystal structures and growalong the [001] direction. This is also confirmed by the contrast of theX-ray diffraction (XRD) pattern which indicates the preferentialorientation of [001] lattice planes in the NBs. The strong and sharp XRDsignals suggest a highly crystalline structure of Ru-LCE NBs, and themain peak at 2.231° corresponds to the preferential [001] growth planeof the Ru-LCE single crystal. According to cell volume and NB size(10000×500×100 nm), a single NB contains about 3.0×10⁸ Ru-LCE molecules.Compared to the usual nanowires with a round cross-section, a nanobeltstructure should provide large area interface when deposited onelectrodes, thus facilitating the fabrication of devices with improvedelectrical contact.

Samples collected 5 min after injection consisted of about 10 nm sizedNPs of amorphous Ru-LCE (see FIG. 3A and FIG. 3B), which subsequentlyaggregated within 30 min into a belt-like structure (see FIG. 3C).Extending the aging time to 2 hours resulted in a progressive increasein the length of the NBs (see FIG. 3D). Electron-diffraction analysisindicated that the initially-formed NB was amorphous Ru-LCE. Extendingthe aging time to 24 h resulted in a progressive increase in thecrystallinity of the NBs. Without being bound by theory, according tothe above observations, formation of Ru-LCE single-crystalline NBsinvolves a multi-step process involving nucleation, oriented assemblyand restructuring of initially formed NP building blocks, rather thandirect growth from solution according to classical mechanisms ofcrystallization.

FIG. 4A shows a comparison of absorption and fluorescence emissionspectra of Ru-LCE NBs and the corresponding monomers in solution. Themonomer in MeCN exhibits a wide intramolecular charge-transferabsorption band (360 to 480 nm), which is assigned to the metal toligand charge transfer (MLCT) transition (that is dπ→π*). Thelowest-energy metal to ligand charge transfer transition of NBs inaqueous solution exhibits an obvious blue-shift (from 480 to 458 nm) ascompared to monomers in acetonitrile, suggesting the formation ofH-aggregates in the NBs due to strong π-stacking interactions. Thefluorescence spectra of the NBs also show a similar hypsochromic shiftas the absorption spectra and an enhanced fluorescence emission (atabout 640 nm), implying that the NBs are J-aggregates, where themolecules are arranged in a head-to-tail direction, inducing arelatively high fluorescence efficiency. The fluorescence image of a NBcan be easily observed (see FIG. 4B). Without being bound by theory, itis believed that the co-existence of two kinds of aggregates (H- andJ-type) leads to spontaneous formation of the NBs.

Two methods were used to observe ECL of the NBs at a platinum electrodeor an ultramicroelectrode (UME). First, ECL from the NBs dispersed inwater containing 0.1 M tripropylamine (TPrA) as a co-reactant and 0.1 Mphosphate buffer could be easily observed during potential scans (from 0to 1.5 V) or pulses (−1.25 to 1.25 V) (see FIG. 5A and FIG. 5B). Second,the ECL curve of a single Ru-LCE NB deposited on a platinum UME was alsoobserved (see FIG. 6A). The cyclic voltammogram (CV) shows a ratherbroad irreversible anodic wave due to the direct oxidation of TPrA, andthe single Ru-LCE NB has little influence on the cyclic voltammogram(CV). The oxidation current begins at about 1.1 V peaks at potential ofabout 1.38 V, and is irreversible. Such anodic oxidation behavior issimilar to that of TPrA in neutral, aqueous solutions at a glassy carbonelectrode. The mechanism likely follows that of Ru(bpy)₃ ²⁺ in solution,where scanning the electrode potential positive of 1.25 V, causes theoxidation of Ru(bpy)₂(bpy-C₁₆Est)²⁺ in the adsorbed NB to the +3 form,either directly, or via TPrA radical cations (illustrated in Schemes IIAand IIB below). Reaction of the +3 form with either the reducing TPrAradical or the +1 form produces the excited state. The ECL emissionintensity increases with increasing potential. Note, that no ECL isobserved for an aqueous solution that is saturated withRu(bpy)₂(bpy-C₁₆Est)²⁺ prepared by putting a slide with a film of thismaterial (cast from an acetonitrile solution) in the solution overnightwith gentle stirring.

Rubrene NCs were prepared by a re-precipitation method as follows: 100μL of 5 mM rubrene solution in tetrahydrofuran (THF) was quicklyinjected into 10 mL of deionized water under an argon atmosphere withvigorous stirring at room temperature. The resulting clear pale red NCsolution was then filtered with a 0.22 μm filter. The hydrodynamicradius of rubrene NPs in water determined by dynamic light scattering(DLS) is about 20 nm and there is a small amount of aggregates around75-100 nm, in size. The kinetics of the formation of the aggregates isdetermined by the interaction between the particles, the particle size,and the flow conditions within system. Addition of capping agents, suchas a low molecular weight surfactant or neutral or charged polymers candramatically affect the formation and/or aggregation process of NPs. Itcan also stabilize the particles. For example, the addition of TritonX-100 in water favors the formation of very small rubrene NCs (˜4 nm),and the size distribution is narrow.

ECL from the rubrene NCs dispersed in water containing 0.1 Mtripropylamine (TPrA) as a co-reactant and 0.1 M phosphate buffer couldbe easily observed during potential scans (from 0 to 0.9 V vs. Ag/AgCl)(see FIG. 7B) or pulses (0 to 1.1 V) (see FIG. 7A, FIG. 7C and FIG. 7D).The ECL signal during the potential pulse is observable but decayssharply with time within the pulse duration (faster than the normaldiffusion-limited mass transfer rate) perhaps due to the instability ofradical ions generated in water medium. The CV in the potential rangeshown in FIG. 7B is featureless, but will reach a diffusion-limitedoxidation peak of TPrA at a positive potential (refer to FIG. 6A).Rubrene NCs prepared in different solvents, such asN,N-dimethylformamide DMF, show much lower ECL signal at the samecondition of preparation, i.e. injection of 100 μL 5 mM rubrene in DMFinto 10 mL of deionized water. Increasing the concentration of rubreneNCs 5 times increases the ECL signal, suggesting that solvent polarityand solubility of the organic or ionic compound in solvents could subtlyaffect the nanocrystallization process in the re-precipitation method.

NCs of DPA were prepared by using THF, DMF or MeCN as a good solvent andwater as a poor solvent. The average hydrodynamic radius of DPA NCsusing MeCN as the dissolving solvent is about 45 nm, which isconsiderably bigger than the rubrene NCs described above. An SEM imageof the DPA NCs reveals that they are polydisperse nanorods withdiameters from about 20 to 100 nm and lengths from about 100-600 nm (seeFIG. 1).

ECL of DPA NCs was examined by comparing TPrA or oxalate as theco-reactant. No significant ECL intensity from DPA NCs was observed byusing TPrA as the co-reactant while some ECL was detected in thepresence of oxalate as shown in FIG. 8. This is consistent withpublished reports, showing that CO₂ ⁻ (the active intermediate producedduring oxalate oxidation) is more energetic than TPrA (the activeintermediate generated from TPrA oxidation). It is believed that thescheme depicted in Scheme 1 may illustrate the scheme by which aco-reactant (e.g., TPrA) may interact with the redox active luminescentcompounds in the present nanostructured materials to generate an excitedstate luminescent species capable of emitting light.

Organic or ionic (e.g., organometallic compounds or metal-ligandcomplexes) nanostructured materials, such as NSMs, have been synthesizedand their ECL has been examined using Ru-LCE, rubrene, and DPA as theindicators and TPrA or oxalate as a co-reactant. The skilled artisanwill recognize that other NPs, co-reactants and other solventcombinations may be used. In order to reduce the background current andenhance the relative ECL efficiency, an electrode or the NPs can undergocertain surface treatments. Suitable electrodes may be formed frommaterials such as ITO, gold, glassy carbon, boron-doped diamond, andother like materials.

In general, “substituted” refers to an organic group as defined below(e.g., an alkyl group) in which one or more bonds to a hydrogen atomcontained therein are replaced by a bond to non-hydrogen or non-carbonatoms. Substituted groups also include groups in which one or more bondsto a carbon(s) or hydrogen(s) atom are replaced by one or more bonds,including double or triple bonds, to a heteroatom. Thus, a substitutedgroup will be substituted with one or more substituents, unlessotherwise specified. In some embodiments, a substituted group issubstituted with 1, 2, 3, 4, 5, or 6 substituents. Examples ofsubstituent groups include: halogens (i.e., F, Cl, Br, and I);hydroxyls; alkoxy, alkenoxy, alkynoxy, aryloxy, aralkyloxy,heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo);carboxyls; esters; ethers; urethanes; oximes; hydroxylamines;alkoxyamines; aralkoxyamines; thiols; sulfides; sulfoxides; sulfones;sulfonyls; sulfonamides; amines; N-oxides; hydrazines; hydrazides;hydrazones; azides; amides; ureas; amidines; guanidines; enamines;imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines;nitro groups; nitriles (i.e., CN); and the like.

Alkyl groups include straight chain and branched alkyl groups havingfrom 1 to about 20 carbon atoms, and typically from 1 to 12 carbons or,in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Alkylgroups further include cycloalkyl groups as defined below. Examples ofstraight chain alkyl groups include those with from 1 to 8 carbon atomssuch as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl,and n-octyl groups. Examples of branched alkyl groups include, but arenot limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl,isopentyl, and 2,2-dimethylpropyl groups. Representative substitutedalkyl groups can be substituted one or more times with substituents suchas those listed above.

Aryl groups are cyclic aromatic hydrocarbons that do not containheteroatoms. Aryl groups include monocyclic, bicyclic and polycyclicring systems. Thus, aryl groups include, but are not limited to,cyclopentadienyl, phenyl, azulenyl, heptalenyl, biphenylenyl, indacenyl,fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl,chrysenyl, biphenyl, anthracenyl, indenyl, indanyl, pentalenyl, andnaphthyl groups. In some embodiments, aryl groups contain 5-14 carbons,and in others from 5 to 12 or even 6-10 carbon atoms in the ringportions of the groups. Although the phrase “aryl groups” includesgroups containing fused rings, such as fused aromatic-aliphatic ringsystems (e.g., indanyl, tetrahydronaphthyl, and the like), it does notinclude aryl groups that have other groups, such as alkyl or halogroups, bonded to one of the ring members. Rather, groups such as tolylare referred to as substituted aryl groups. Representative substitutedaryl groups can be mono-substituted or substituted more than once. Forexample, monosubstituted aryl groups include, but are not limited to,2-, 3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups, which can besubstituted with substituents such as those listed above.

Heterocyclyl groups include aromatic (also referred to as heteroaryl)and non-aromatic ring compounds containing 3 or more ring members, ofwhich one or more is a heteroatom such as, but not limited to, N, O, andS. In some embodiments, heterocyclyl groups include 3 to 20 ringmembers, whereas other such groups have 3 to 6, 3 to 10, 3 to 12, or 3to 15 ring members. Heterocyclyl groups encompass unsaturated, partiallysaturated and saturated ring systems, such as, for example, imidazolyl,imidazolinyl and imidazolidinyl groups. The phrase “heterocyclyl group”includes fused ring species including those comprising fused aromaticand non-aromatic groups, such as, for example, benzotriazolyl,2,3-dihydrobenzo[1,4]dioxinyl, and benzo[1,3]dioxolyl. The phrase alsoincludes bridged polycyclic ring systems containing a heteroatom suchas, but not limited to, quinuclidyl. However, the phrase does notinclude heterocyclyl groups that have other groups, such as alkyl, oxoor halo groups, bonded to one of the ring members. Rather, these arereferred to as “substituted heterocyclyl groups”. Heterocyclyl groupsinclude, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl,imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl,tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl,imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl,thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, morpholinyl,thiomorpholinyl, tetrahydropyranyl, tetrahydrothiopyranyl, oxathiane,dioxyl, dithianyl, pyranyl, pyridyl, bipyridyl, pyrimidinyl,pyridazinyl, pyrazinyl, triazinyl, dihydropyridyl, dihydrodithiinyl,dihydrodithionyl, homopiperazinyl, quinuclidyl, indolyl, indolinyl,isoindolyl, azaindolyl (pyrrolopyridyl), indazolyl, indolizinyl,benzotriazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl,benzthiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiinyl,benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl,benzothiadiazolyl, benzo[1,3]dioxolyl, pyrazolopyridyl, imidazopyridyl(azabenzimidazolyl), triazolopyridyl, isoxazolopyridyl, purinyl,xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, quinolizinyl,quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl,pteridinyl, thianaphthalenyl, dihydrobenzothiazinyl,dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl,tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl,tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl,tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl,tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups.Representative substituted heterocyclyl groups can be mono-substitutedor substituted more than once, such as, but not limited to, pyridyl ormorpholinyl groups, which are 2-, 3-, 4-, 5-, or 6-substituted, ordisubstituted with various substituents such as those listed above.

Alkoxy groups are hydroxyl groups (—OH) in which the bond to thehydrogen atom is replaced by a bond to a carbon atom of a substituted orunsubstituted alkyl group as defined above. Examples of linear alkoxygroups include but are not limited to methoxy, ethoxy, propoxy, butoxy,pentoxy, hexoxy, and the like. Examples of branched alkoxy groupsinclude but are not limited to isopropoxy, sec-butoxy, tert-butoxy,isopentoxy, isohexoxy, and the like. Examples of cycloalkoxy groupsinclude but are not limited to cyclopropyloxy, cyclobutyloxy,cyclopentyloxy, cyclohexyloxy, and the like. Representative substitutedalkoxy groups can be substituted one or more times with substituentssuch as those listed above.

Illustrative Embodiments

While the making and using of various embodiments of the presentinvention are discussed in detail herein, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

One embodiment provides a nanostructured particulate material formedfrom a redox active luminescent compound. The nanostructured materialcommonly has a least one dimension which has an average size no largerthan about 250 nm, more commonly no more than about 150 nm and, in someembodiments, at least one dimension may have an average size no largerthan about 100 nm. For example, the nanostructured material may be ananoparticle (“NP”) in which no dimension has an average size largerthan about 200 nm. Other examples include nanocrystals (“NCs”) in which,typically, at least two and, often three, dimensions are no more thanabout 200 nm and commonly no more than about 100 nm. Other embodimentsmay include nanobelts (“NBs”), which have long, straight and belt-likemorphology, with a width of at least about 150 nm and a thickness ofabout 50 to 125 nm. Such nanobelts may have widths of about 200 to 1000nm and lengths of about 5 to 15 μm, and typically have awidth-to-thickness ratio of about 5 to 10 and may have an aspect ratioof about 10 or more. In still other embodiments, the nanostructuredmaterial may be a nanorod having an average diameter of no more thanabout 250 nm, commonly about 10 to 150 nm, and an average length ofabout 50 nm to 1 micron.

One embodiment provides a method for detecting an analyte in a samplesolution to which luminescent nanostructured particulate materials havebeen added. The method includes contacting the sample solution with anelectrochemical cell containing two or more electrodes in communicationwith the solution; and generating one or more ECL properties through aninteraction of the luminescent nanostructured materials, the liquidsample and two or more of the electrodes; and measuring at least one ECLproperty generated by the interaction. The luminescent nanostructuredmaterials include a redox active luminescent organic or organometalliccompound.

Another embodiment provides a method of determining the presence of ananalyte of interest comprising:

-   -   (a) contacting the analyte with a chemical moiety under suitable        conditions so as to form a reagent mixture; wherein the chemical        moiety includes a nanostructured particulate material comprising        a redox active luminescent organic or organometallic compound;    -   (b) inducing the chemical moiety to emit electromagnetic        radiation by exposing the reagent mixture to chemical or        electrochemical energy; and    -   (c) detecting the emitted electromagnetic radiation and thereby        determining the presence of the analyte of interest.

Suitable redox active luminescent compounds, which can be used to formthe present nanostructured materials, can be selected from a variety oforganic and/or ionic luminescent compounds. Examples include luminescentaromatic hydrocarbons, e.g., luminescent phenyl substituted aromatichydrocarbons such as phenyl substituted polycyclic aromatic compounds,and luminescent metal-containing complexes, e.g., heteroaromaticpolydentate complexes of a metal ion such as ruthenium.

Examples of suitable phenyl substituted polycyclic aromatic compounds,often in the form of nano structured luminescent materials (which may bein the form of nanocrystals or nanorods), include rubrene (“Rub”),diphenylanthracene (“DPA”) and other luminescent phenyl substitutedpolycyclic aromatic compounds. Heteroaromatic polydentate complexesinclude aromatic compounds with one or more heteroatoms in which thecomplex may bind more than one metal or may bind more than once to asingle metal.

Examples of suitable luminescent metal-containing complexes includepolydentate complexes of a metal ion such as ruthenium, osmium, rhenium,cerium, europium, terbium, and/or ytterbium. Particular examples ofsuitable heteroaromatic polydentate complexes include a substitutedbis(2,2′-bipyridyl)ruthenium(II) or tris(2,2′-bipyridyl)ruthenium(II)containing moieties, wherein at least one of the bipyridyl (“bpy”)groups is substituted with one or more long chain alkyl groups.

Suitable long chain alkyl substituted bpy groups (“LCsub-bpy”) include:4-methyl-4′-alkanoylaminomethyl-2,2′-bipyridines;4,4′-bis-(alkoxymethyl)bipyridines;4,4′-bis-(alkylmercaptomethyl)bipyridines; alkyl esters ofomega-(4-methyl-2,2-bipyridine-4′-yl)-alkanoic acids; di-n-alkyl4,4′-bis-(carboxylate)bipyridines; n-alkyl diesters of4,4′-bis-(carboxy)-2,2-bipyridines; and diesters of long chain fattyacid with 4,4′-bis-(hydroxymethyl)bipyridines.

Examples of suitable luminescent metal complexes, including n-alkyldiesters of 4,4′-bis-(carboxy)-2,2-bipyridines, include Ru(II)bis(2,2′-bipyridine)(di-n-alkyl 4,4′-bis-(carboxylate)bipyridine)²⁺salts, e.g., Ru(II) bis(2,2′-bipyridine)(di-n-pentadecyl4,4′-bis-(carboxylate)bipyridine)²⁺ salts or other related rutheniumcomplexes, where the alkyl ester group each contain about 10 to 25carbon atoms. Suitable examples of such alkyl ester groups include suchesters of stearyl alcohol, palmityl alcohol and dodecyl alcohol.

Examples of suitable luminescent metal complexes based on4-methyl-4′-alkanoylaminomethyl-2,2′-bipyridines include Ru(II)bis(2,2′-bipyridine)(4-methyl-4′-alkanoylaminomethyl-2,2′-bipyridine)²⁺salts, e.g., Ru(II)bis(2,2′-bipyridine)(4-methyl-4′-stearoylaminomethyl-2,2′-bipyridine)²⁺salts.

Examples of suitable luminescent metal complexes based on4,4′-bis-(alkoxymethyl)bipyridines include Ru(II)bis(2,2′-bipyridine)(4,4′-bis-(n-alkoxymethyl)bipyridine)²⁺ salts e.g.,Ru(II) bis(2,2′-bipyridine)(4,4′-bis-(n-hexadecyloxymethyl)bipyridine)²⁺salts.

Examples of suitable luminescent metal complexes based on4,4′-bis-(alkyl mercaptomethyl)bipyridines include Ru(II)bis(2,2′-bipyridine)(4,4′-bis-(n-alkylmercaptomethyl)bipyridine)²⁺ saltse.g., Ru(II)bis(2,2′-bipyridine)(4,4′-bis-(n-alkylmercaptomethyl)bipyridine)²⁺salts.

Other examples of suitable luminescent metal complexes based on alkylesters of omega-(4-methyl-2,2-bipyridine-4′-yl)-alkanoic acids includeRu(II) bis(2,2′-bipyridine)(4-methyl-2,2-bipyridine-4′-yl)-alkanoic acidalkyl ester)²⁺ salts, e.g., Ru(II)bis(2,2′-bipyridine)-4-(4-methyl-2,2-bipyridine-4′-yl)-butyric aciddecyl ester)²⁺ salts. Additional examples include Ru(II)bis(2,2′-bipyridine)(di-n-alkanoyl ester of4,4′-bis-(hydroxymethyl)bipyridine)²⁺ salts, e.g., Ru(II)bis(2,2′-bipyridine)(di-stearoyl ester of4,4′-bis-(hydroxymethyl)bipyridine)²⁺ salts; and Ru(II)bis(2,2′-bipyridine)(di-palmitoyl ester of4,4′-bis-(hydroxymethyl)bipyridine)²⁺ salts.

Another embodiment provides a method for detecting the presence of ananalyte of interest in a liquid sample, the method comprising:

(a) contacting the sample with a reagent comprising a nanostructuredmaterial; wherein the reagent is capable of being induced toelectrochemiluminesce repeatedly and the nanostructured materialcomprises a redox active luminescent organic and/or ionic compound;

(b) inducing the reagent to electrochemiluminesce repeatedly; and

(c) detecting the presence of luminescence emitted thereby detecting thepresence of the analyte of interest in the sample. The method may alsoinclude contacting the sample with the reagent and an ECL coreactant,such as such as an oxalate salt (e.g., sodium oxalate) or atrialkylamine (e.g., tripropylamine). For example, in many embodimentsof the method, the nanostructured material may include a polydendatemetal complex (such as a ruthenium bipyridyl complex) and trialkylaminecoreactant (e.g., tripropylamine). In other embodiments of the method,the nanostructured material may include a phenyl substituted polycyclicaromatic hydrocarbon (such as rubrene) and trialkylamine coreactant(e.g., tripropylamine). In still other embodiments of the method, thenanostructured material may include a phenyl substituted polycyclicaromatic hydrocarbon (such as diphenylanthracene) and an oxalate saltcoreactant (e.g., sodium oxalate).

Another embodiment provides a method for quantitatively determining theamount of an analyte of interest present in a liquid sample, the methodcomprising: (a) contacting the sample with a reagent comprising ananostructured material; wherein the reagent is capable of being inducedto electrochemiluminesce repeatedly; (b) inducing the reagent toelectrochemiluminesce repeatedly; and (c) determining the amount ofluminescence emitted and thereby quantitatively determining the amountof the analyte of interest present in the sample. The nanostructuredmaterial typically includes a redox active luminescent organic and/orionic compound. The method may also include contacting the sample withthe reagent and an ECL coreactant, such as such as sodium oxalate ortrialkylamine (e.g., tripropylamine).

Another embodiment provides a method for detecting the presence of ananalyte of interest in a liquid sample, the method comprising:

(a) contacting the sample with a reagent comprising a nanostructuredmaterial; wherein the reagent is capable of being induced toelectrochemiluminesce repeatedly and the nanostructured materialcomprises a redox active luminescent organic and/or organometalliccompound;

(b) inducing the reagent to electrochemiluminesce repeatedly; and

(c) detecting the presence of luminescence emitted thereby detecting thepresence of the analyte of interest in the sample. The method may alsoinclude contacting the sample with the reagent and an ECL coreactant,such as an oxalate salt (e.g., sodium oxalate) or trialkylamine (e.g.,tripropylamine).

Another embodiment provides a method for quantitatively determining theamount of an analyte of interest present in a liquid sample, the methodcomprising: (a) contacting the sample with a reagent comprising ananostructured material; wherein the reagent is capable of being inducedto electrochemiluminesce repeatedly; (b) inducing the reagent toelectrochemiluminesce repeatedly; and (c) determining the amount ofluminescence emitted and thereby quantitatively determining the amountof the analyte of interest present in the sample. The nanostructuredmaterial typically includes a redox active luminescent organic and/orionic compound. The method may also include contacting the sample withthe reagent and an ECL coreactant, such as such as sodium oxalate ortrialkylamine (e.g., tripropylamine).

Another embodiment provides a method of determining the presence of ananalyte of interest in a sample comprising:

-   -   (a) contacting the sample with a chemical moiety under suitable        conditions so as to form a reagent mixture; wherein the chemical        moiety includes a nanostructured particulate material comprising        a redox active luminescent compound;    -   (b) inducing the chemical moiety to emit electromagnetic        radiation; and    -   (c) detecting the emitted electromagnetic radiation and thereby        determining the presence of the analyte of interest;

wherein inducing the chemical moiety to emit electromagnetic radiationcomprises exposing the reagent mixture to chemical, electrochemicaland/or electromagnetic energy; and

the redox active luminescent compound includes a luminescent polycyclicaromatic hydrocarbon, such as a phenyl substituted polycyclic aromatichydrocarbon. The reaction mixture may also include an ECL coreactant,such as sodium oxalate, persulfate, benzoyl peroxide, or a trialkylamine(e.g., tripropyl amine).

The nanostructured material comprising luminescent polycyclic aromatichydrocarbon may be in the form of redox active, luminescentnanoparticles having an average hydrodynamic radius of no more thanabout 100 nm. Such nanoparticles may be formed from a phenyl substitutedpolycyclic aromatic hydrocarbon such as rubrene. For example,nanocrystals formed from phenyl substituted polycyclic aromatichydrocarbons may have a hydrodynamic radius of no more than about 50 nm(as determined as a dispersion in water determined by dynamic lightscattering (DLS)) which may also include a small amount of nanocrystalaggregates around 75-100 nm in size. In other embodiments, thenanostructured material comprising luminescent polycyclic aromatichydrocarbon may be in the form of nanorods, e.g., nanorods having adiameter of about 10 to 150 nm and a length of about 50 nm to 1 micron.Such nanorods maybe formed from a phenyl substituted polycyclic aromatichydrocarbon such as diphenylanthracene. nanorods maybe formeddiphenylanthracene may have a diameter of about 20 to 100 nm and alength of about 100 nm to 600 nm.

A method of determining the presence of an analyte of interest in asample comprising:

-   -   (a) contacting the sample with a chemical reagent under suitable        conditions so as to form a reagent mixture; wherein the chemical        reagent includes a nanostructured particulate material        comprising a redox active luminescent compound;    -   (b) inducing the chemical reagent to emit electromagnetic        radiation; and    -   (c) detecting the emitted electromagnetic radiation and thereby        determining the presence of the analyte of interest;

wherein inducing the chemical reagent to emit electromagnetic radiationcomprises exposing the reagent mixture to chemical, electrochemicaland/or electromagnetic energy; and

the redox active luminescent compound includes a polydendate metalcomplex, such as a bipyridyl containing metal complex. The reactionmixture may also include an ECL coreactant, such as oxalate salt,persulfate salt, benzoyl peroxide, or a trialkylamine (e.g., tripropylamine).

The nanostructured particulate material comprising the redox activeluminescent compound may includes a redox active, luminescentpolydendate metal complex, such as a luminescent heteroaromaticpolydendate metal complex. Examples of suitable luminescentheteroaromatic polydendate metal complexes may include a ruthenium,osmium, rhenium, cerium, europium, terbium and/or ytterbium ion.Suitable nanostructured materials comprising polydendate metal complexesinclude luminescent nanobelts formed from heteroaromatic polydendatemetal complexes containing one or more long chain alkyl substitutedligands. One examples of such nanostructured materials are nanobeltsformed from heteroaromatic polydendate ruthenium complexes which includeat least one long chain alkyl substituted bipyridine ligand. Suchnanobelts can have widths of about 200 to 1000 nm and lengths of about 5to 15 μm. The thickness of these nanobelts can range from around 50 to120 nm (as characterized by field-emission scanning electronmicroscopy).

Other embodiments are directed to methods which employ nanostructuredparticulates formed from redox active, luminescent polycyclic aromatichydrocarbon. Such nanostructured particulates commonly have a least onedimension which has an average size no larger than about 250 nm and, insome instances, no larger than about 100 nm.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

While the compositions and methods of this invention have been describedin terms of preferred embodiments, it will be apparent to those of skillin the art that variations may be applied to the compositions and/ormethods and in the steps or in the sequence of steps of the methoddescribed herein without departing from the concept, spirit and scope ofthe invention. All such similar substitutes and modifications apparentto those skilled in the art are deemed to be within the spirit, scopeand concept of the present invention.

1. A method of determining the presence of an analyte of interest in asample comprising: (a) forming a reagent mixture, which comprises achemical reagent and the sample; wherein the chemical reagent comprisesluminescent nanostructured material formed from a redox active,luminescent compound; (b) inducing the luminescent nanostructuredmaterial to emit electromagnetic radiation; and (c) detecting theemitted electromagnetic radiation; wherein inducing the luminescentnanostructured material to emit electromagnetic radiation comprisesexposing the reagent mixture to chemical and/or electrochemical energy.2. The method of claim 1, wherein inducing the luminescentnanostructured material to emit electromagnetic radiation comprisesinducing the luminescent nanostructured material toelectrochemiluminesce repeatedly; and (c) detecting the emittedelectromagnetic radiation comprises detecting the presence of theemitted luminescence.
 3. The method of claim 1, wherein inducing theluminescent nanostructured material to emit electromagnetic radiationcomprises exposing the reagent mixture to electrochemical energy.
 4. Themethod of claim 1, wherein the reagent mixture further comprises an ECLcoreactant.
 5. The method of claim 1, wherein the luminescentnanostructured material comprises luminescent nanoparticles formed froma luminescent polycyclic aromatic hydrocarbon.
 6. The method of claim 1,wherein the luminescent nanostructured material comprises luminescentnanoparticles formed from a redox active, ionic luminescent compound. 7.The method of claim 6, wherein the redox active, ionic luminescentcompound comprises a luminescent polydendate metal complex.
 8. Themethod of claim 6, wherein the redox active, ionic luminescent compoundcomprises a luminescent, heteroaromatic polydendate ruthenium complex.9. The method of claim 1, wherein the luminescent nanostructuredmaterial comprises luminescent nanobelts formed from a luminescentpolydendate metal complex, which includes at least one long chain alkylsubstituted bipyridine ligand.
 10. The method of claim 1, wherein theluminescent nanostructured material comprises nanoparticles formed froma redox active, luminescent phenyl substituted polycyclic aromatichydrocarbon; wherein the nanostructured particulates have a least onedimension which is no larger than about 250 nm and can be induced torepeatedly emit electromagnetic radiation by direct exposure to anelectrochemical energy source.
 11. The method of claim 10, wherein thenanoparticles are substantially free of surfactant.
 12. The method ofclaim 10, wherein the nanoparticles have an average hydrodynamic radiusof no more than about 100 nm.
 13. The method of claim 10, wherein thephenyl substituted polycyclic aromatic hydrocarbon is rubrene.
 14. Themethod of claim 10, wherein the phenyl substituted polycyclic aromatichydrocarbon is 9,10-diphenylanthracene.
 15. The method of claim 1,wherein the luminescent nanostructured material comprises nanoparticlesformed from a redox active, ionic luminescent compound; wherein thenanoparticles have a least one dimension which is no larger than about250 nm.
 16. The method of claim 15, wherein the redox active, ionicluminescent compound comprises a heteroaromatic polydendate metalcomplex.
 17. The method of claim 16, wherein the heteroaromaticpolydendate metal complex comprises at least one long chain alkylsubstituted bipyridyl ligand.
 18. The method of claim 16, wherein theheteroaromatic polydendate metal complex comprises a long chain alkylsubstituted Ru(bpy)₃ ²⁺ complex.
 19. The method of claim 16, wherein theheteroaromatic polydendate metal complex comprises a ruthenium, osmium,rhenium, cerium, europium, terbium, and/or ytterbium ion.
 20. The methodof claim 16, wherein the heteroaromatic polydendate metal complex is aluminescent, heteroaromatic polydendate ruthenium complex.
 21. Themethod of claim 15, wherein the nanostructured particulates compriseluminescent nanobelts formed from a luminescent polydendate metalcomplex, which includes at least one long chain alkyl substitutedbipyridine ligand.
 22. The method of claim 21, wherein the luminescentnanobelts are formed from a luminescent, heteroaromatic polydendateruthenium complex.
 23. The method of claim 21, wherein the luminescent,heteroaromatic polydendate ruthenium complex comprises a long chainalkyl substituted Ru(bpy)₃ ²⁺ complex.
 24. The method of claim 1,wherein inducing the luminescent nanostructured material to emitelectromagnetic radiation comprises inducing the luminescentnanostructured material to electrochemiluminesce repeatedly; and (c)detecting the emitted electromagnetic radiation comprises detecting thepresence of the emitted luminescence; the reagent mixture furthercomprises a trialkyl amine ECL coreactant; and the luminescentnanostructured material comprises nanoparticles of a luminescent,heteroaromatic polydendate ruthenium complex having a least onedimension which is no larger than about 250 nm.
 25. The method of claim24, wherein the luminescent, heteroaromatic polydendate rutheniumcomplex is a long chain alkyl substituted Ru(bpy)₃ ²⁺ complex.